For Li Hua

Ranganathan, P.N. via (by pnr from
Mon Jun 18 07:51:50 EST 2007

Protein purification is not 1-size fits all, due to various factors
influencing folding.

Is the his-tag in the C-terminus? That will help you in getting only
full-length proteins, upon Ni-column chrom.


If you know the amino acid sequence, use the tools in the protein
databases/sources such as PDB,  "ExPASY" to assess its biochemical
parameters. This would help in purification scheme.


Since you know the correct band, estimate its MWr from SDS-PAGE, use
gel-filtration column chromatography, in the presence of appropriate
detergent and pH. Select a resin with a correct "fractionation range" and a
pore-size that would enable your protein to be roughly in the middle of the
elution profile. Make sure that the resin is compatible with your
conditions. Then concentrate the eluate. Maintain buffer, ionic strength,
pH, detergent or chaotropic agent conditions.


It is a big assumption that a fully synthesized (therefore folded) protein,
would yield a conformation upon denaturation-renaturation identical to a
never-denatured protein. This is so because of short-contacts and long-range
interactions between residues, among others. Assuming that it is:  Do not
"over-express". Stop the culture when your protein concentration in
bacterial extract is low. Culture more flasks for more yield. This way, it
MAY not localize in the inclusion bodies in the first place. Then you get to
work with a "native" protein.


Hope it helps. Have fun.


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